Scavenger packet



Feb. 14, 1961 R. R. BARTON SCAVENGER PACKET Filed NOV. 25, 1958 643-PERMEABLE MEMBRANE "WA 7ER-IMPERMEABLQ IN V EN TOR.

RICHARD [2. BAR TON A TTORNEV United States Patent Ofii 2,971,850Patented eh. 14, 1

SCAVENGER PACKET Richard R. Barton, Waldwick, N.J., assignor to MilesLaboratories, Inc., Elkhart, Ind., a corporation of Indiana Filed Nov.25, 1958, Ser. No. 776,388 11 Claims. (Cl. 99-171) This inventionrelates to a deoxygenating process and to a product useful therein.Particularly, the invention relates to a deoxygenating body and themethod of its use. More particularly, the invention relates to adeoxygenating body, or scavenger packet, which comprises an enzymesystem having glucose oxidase activity, a substrate for said enzymesystem and a liquid, the components of said body being confined in awater-impermeable, gas-permeable membrane in such fashion that one ofthe components is separated from the other components by a rupturableseal.

Those familiar with the art of enzyme chemistry are aware of enzymaticprocesses for the removal of oxygen or glucose from food or otherproducts through the use of an enzyme system having glucose oxidaseactivity. The reaction upon which these processes are based proceeds inaccordance with the following schematic diagram:

glucose glucose-l-Ori-H O gluconie acid-l-HzOz oxidase .catalase 21120:2 20+ 9 One such process is described and claimed in US. Reissue PatentNo. 23,523, issued to Dwight L. Baker, July 22, 1952. The principle ofbasic patent is referred to in US. Patent No. 2,758,932, issued August14, 1956, to Don Scott.

The Scott patent teaches a method of deoxygenating a hermetically sealedcontainer by means of a deoxygenating body comprising a dispersion ofwater, glucose, and an enzyme system having glucose oxidase activity. Inthis patent the components of the deoxygenating body are separated fromthe contents of the container by an oxygen permeable, moisture-proofbarrier such as cellophane, wax paper, and the like.

The instant invention is closely related to the packet of the Scottpatent, but, as will be pointed out below, represents an improvementthereon.

It has been found that even under the most carefully controlledconditions a deoxygenating body prepared in accordance with theteachings of the Scot patent, that is, when the components of thedeoxygenating body are in contact with each other within the membraneenvelope, becomes substantially inactive and loses it ability to take upoxygen after the lapse of short periods of time if exposed to theatmosphere. Preparation of these prior art packets in the absence ofatmospheric oxygen, and maintaining them under a nitrogen blanket untiluse ha proven impractical. Attempts have been made to package the drycomponents and inject the requisite liquid immediately prior to use bysome means such as a hypodermic needle, but this expedient also provedto be unsatisfactory on a commercial scale.

It has now been found, however, and forms the object of this invention,that the above disadvantages may be overcome if a deoxygenating bodycomprising a liquid, an enzyme system having glucose oxidase activity,and a substrate for said enzyme is enclosed in a packet of twoccmpartments separated by a rupturable seal, one com-. partmentcontaining one of the three components of the body and the othercompartment containing the other two. In such a device no reaction cantake place since all of the components must be in intimate contactbefore the enzyme will oxidize the substrate. The two compartments areseparated by a rupturable seal which breaks under pressure allowing thethree components to mix and thus activating the deoxygenating body. Suchrupture is accomplished immediately prior to use of the packet and thusthe packet has all its activity preserved to the time of use.

Fig. 1 represents a schematic drawing of one embodiment of the scavengerpacket of this invention. Fig. 2 represents an end view of the packet ofFig. 1. Fig. 3 represents a second embodiment of the inventive concept,and Fig. 4 represents an end view of the packet of Pi 3.

lurning now to the drawing, the packet or envelope of the figures areconstructed of a gas-permeable, water-impermeable membrane such aspolyethylene, laminated polyethylene, cast tefion, pliofilm, styrene,cellophane, and the like. The material must be gas-permeable to permitthe passage of oxygen or oxygen containing gas such as air, andwater-impermeable to prevent the passage of liquid. It will beunderstood that in certain embodiments of the invention, such as thatshown in Fig. 3, that one compartment of the packet may be composed of agas and liquid impermeable film. It is essential, however, that agas-permeable liquid-impermeable film separate the total mixture fromthe surrounding area in order that oxygen containing gas may passthrough the film and react in accordance with the equation set outabove.

The packet of Figures 1 and 2 is constructed so that there is presenttwo compartments 2 separated by a rupturable seal 4. The other edges ofthe compartments 6 are heat-sealed by methods known to the art.

As was stated above, the deoxygenating body enclosed within the packetcomprises an enzyme having glucose oxidase activity, a substrate forthat enzyme and a liquid. In the packet of Figures 1 and 2, two of thesethree components are confined within one of the compartments 2 and theother component is confined in the other compartment. For example, theenzyme may be confined in one compartment and the substrate and theliquid in the other; the enzyme and the liquid may be mixed and placedin one compartment and the substrate in the other; or the enzyme andsubstrate confined in one compartment and the liquid in the other. It isessential that the components of the deoxygenating body e kept separatedprior to use to prevent deactivation of the deoxygenating body. Thepacket is activated immediately prior to use by breaking the rupturableseal 4 by passing the packet through low clearance rollers, or otherobvious means, thus allowing the three components to become admixed.Oxygen or an oxygen-containing gas passes through the membrane and isutilized in the reaction, thus completely deoxygenating the air-spacearound the packet.

For use in vacuum packaging, it may be desirable that a pinhole beplaced in each compartment of the packet to permit rapid passage of airand the concomitant equalization of pressure at the time of vacuumapp1ica tion. This prevents rupture of the packet due to the suddenpressure differential caused by the vacuum application during vacuumpacking.

The utility of the packet of the invention is obvious; one or more, asdesired, may be activated and placed in a container of material subjectto oxidative deterioration and the'container hermetically sealed. Theoxygen in the container is consumed by the system and the contents ofthe container remain free of oxidative deterioration as long as the sealremains unbroken.

As has been previously stated, the components of the 'deoxygcn'atingbody comprise a liquid, an enzyme system having glucose oxidaseactivity, and a substrate for said enzyme system. The liquid componentis preferably water, although other liquids which do not impair theactivity of the'enzyme system may be used. Other substances may beincluded, such as enzyme activators, humecta'nts, extenders, and thelike. Such substances include aqueous acetate or citrate buffer, aqueoussodihm'gluconate, aqueous glycerine, dilute ethanol and the like.

Enzyme systems having glucose 'oxidase activity have as their primaryand principal constituent the enzyme "glucose oxidase. One such enzymesystem which also contains catalase and which is commercially availablecontains 1500 glucose oxidase units per gram, a unit being "defined asthat amount which will take up 10 cubic millimeters of oxygen per minuteunder the following conditions: 3.3% cerelose, 0.4% sodium salt ofdehydroactetic acid, M/ 10 phosphate butler pH 5.1 and temperature 30 C.This enzyme system also contains up to about 1100 units of catalase pergram, a unit being defined as that amount of catalase which willdecompose 300 mg. of H under standard conditions. As is well known, the'catalase breaks down hydrogen peroxide to water and molecular oxygen.

The substrate for the enzyme system will, of course, contain glucose.Preferably the substrate contains a major amount of glucose such as ispresent in cerelose, dextrose, corn syrup, etc.

Since the enzyme system operates most efficiently at a pH of'between 4.5and 8.0, and since gluconic acid is one :product of the reaction, it ispreferred to have present a bufiering agent in an amount sufficient tomaintain the pH of the activated packet within the optimum pH range.Suitable buffers include alkali metal phosphates, calcium carbonate,sodium acetate, sodium citrates and the like.

It is preferred that the substrate for the enzyme system be thoroughlyadmixed with a filler material to increase surface area to speed theoxygenation reaction and :to assist in preventing the liquid fromdiffusing through theliquid impermeable film. The filler, preferablyselected from the group of ground balsa wood pulp, cereal products, finesawdust, carboxymethyl cellulose, agar, gelat n, gum, silica gel,cliatomaceous earth, etc., prevents the liquid from gasifying andescaping through the gaspermeable membrane.

It 1 s desired that there be present in the formulation a stabilizermaterial in order to insure against loss of enzyrne potency. Thisstabilizer material may comprise s lica gel, glycerine or similarmaterials. It will normally be present in amounts varying between about0.10 to 0.33 g. per ml. of solution, preferably from about 0.15 to about0.25 gm./ml.

It has been found advantageous to include in the formulation apreservative material in order to assist the stabilizer in insuringagainst loss of enzyme potency. Thispreservative may be selected fromsodium gluconate solution, a solution of other salts of food acid andthe like. Although it has been found that from 0.10 to 0.50 ml. of asodium gluconate having a molarity between 0.38 and 1.5 is operable, itis preferred to use from 0.25 to 0.35 ml. of a sodium gluconate solutionhaving molarity of 0.75 per packet.

It will be understood, of course, that difie'rent amounts 5 above thatmoisture content of the packet had a deleteof the components of thedeoxygenating body will be used depending upon the amount of oxygen tobe consumed.

Ranges of operable and preferred proportions are set outin tabular formbelow:

Range of components Operable Preferred Enzyme system (units) 100-550200-400 Substrate:

Glucose (gms) 1130-2. 00 0. -1. 50 Stabilizer (mg./ml.) 0. 10-0. 330.15-0. 2a Preservative (g. 0.10-0.50 '0. 25-035 Filler (gum) 0. 10-1.00 0. 30-0. 60 Buffer (gun) 0. 01-0. 30 0018-01200 Water (mL) 1. 00-3.00 1.80-2.20

The invention described in general terms above Will be more specificallyexplained by reference to the following; illustrative examples:

EXAMPLE I Scavenger packets in accordance with the inventive conceptwere prepared usingv thefollowing formulation as the dry component:

Glucose oxida'se 2.0,lbs. Cerelose -1. 400 lbs. Filter aid('diatomaceous'earth) 7 lbs. 9 ozs. Calcium carbonate 1.0 lbs. Sodiumacetate 1 lb. 14 ozs. Citric acid .2 lbs."'8 ozs.

TABLE I Efiect of temperature on oxygen pickup Mm. O2 consumed 48 hours.108.h0u1s EXAMPLE II.

Efiect of inois i ui'e On oxygl P Percent Mm. 02124 I120. hours absorbedEXAMPLE HI Since it was apparent from the data in Example 11 riouseffect upon the rate of oxygen uptake, the packets of the followingformulation were prepared:

TABLE III Grn. Mm. Or pickup Balsa Wood EXAMPLE IV Glucose oxidase wasdissolved in a sodium gulconate solution of varying molarity and dilutedwith 30% glycerine to a potency of 150 glucose oxidase units per m1. Two(2) ml. of this liquid was placed in one compartment of a 1 milpolyethylene packet and in the other compartment was placed 1.5 g. orthe following formulations:

Grams Cerelose 145.8 Silica gel 10.0 Calcium carbonate 4.0 Sodiumacetate 6.9 Citric acid 9.3 Balsa wood .5 Sodium gluconate Variable Theefiect of varying the amount of the sodium gluconate in the formulationon oxygen pickup is set out in Table IV below.

TABLE IV Molarity Mm. O:

Na gluconsumed conate 24 hours EXAMPLE V The liquid component of thepacket of Example IV containing varying amounts of sodium gluconate wasvtested for stability over a 30 day period. Results of this stabilitytest in glucose oxidase units per ml. are set out in Table V below.

TABLE V Potency (u./ml.) Molarity Na gluconate Original After 1 6EXAMPLE VI The scavenger packets of Example IV were activated andimmediately sealed in No. 2 cans and the oxygen uptake at the end of 24hours was measured. The data obtained are set out in Table VI below.

TABLE VI Molarity Mm. 02

sodium consumed gluconate EXAMPLE VII In one compartment of a 1 milpolyethylene packet prepared in accordance with the concept of thisinvention there was placed 1.3 g. of cerelose and 0.2 g. of citric acid.In a similar packet there was placed 1.3 g. of cerelose, 0.2 g. ofcitric acid and 0.5 g. of ground balsa wood. In the other compartment ofeach packet there was placed 2 ml. of a glucose oxidase solutionprepared as described in connection with Example IV above. The packetswere sealed, the rupturable seal broken immediately prior to testing andthe packets placed in No. 2 cans which were hermetically sealed. At theend of 24 hours the packet without the balsa Wood consumed 10,810 mm. 0whereas the packet containing the balsa wood consumed 26,200 mm. O

EXAMPLE VIII TABLE VII Citric Acid Mm. Oz

(mgm) consumed EXAMPLE IX In one compartment of a 1 mil polyethylenepacket there was placed 1.8 g. of a dry mix having the followingformula:

Parts Cerelose Citric acid 2 Balsa wood 50 In the other compartmentthere was placed 2.0 ml. of a liquid glucose oxidase compositionprepared as described in Example IV above. The packets were sealed andthe rupturable seal broken immediately before placing in No. 2 cansfilled with a standard volume of a white cake mix. Similar packets wereplaced in a No. 2 can containing walnut meats. After the expiration ofone week the percentage of oxygen remaining in the head space wasmeasured.

In the case of white cake mix, those cans containing scavenger packetswere found to contain 9.5% oxygen. Control cans without scavengerpackets contained 20.3% oxygen. In the case of the walnut meats thosecans containing the scavenger packets contained 10.5% oxygen;

the liquid component calculated.

Parts Cerelose 130 Citric acid 2 Balsa wood 50 In the other compartmentthere was placed 2.0 m1. of

a liquid glucose oxidase composition prepared as described in Example IVabove.

The packets were weighed and held at room temperature and room humidityfor 72,hours. At the end of that period they were reweighed and themoisture loss of It was found that at the end of 72 hours the moistureloss amounted to 19.7 mg. (0.07%).

At the end of 5 /2 hours the oxygen uptake was measured to be 12,500 cu.mm. At the end of 72 hours the oxygen uptake was 19,500 cu. mm.

EXAMPLE XI Using a polyethylene having a thickness of 0.50 mil, packetswere prepared as described in connection with Example X above. Thesepackets were placed in a desiccator over calcium chloride for one weekand lost 2.57% of the moisture of the liquid component during that time.

Packets prepared from this polyethylene were measured for oxygen uptakeat the end of 23 hours. The oxygen uptake was found to be 56,400 cu. mm.

EXAMPLE. XII Using the components as described in connection with thepackets of Example X above, but using cast Teflon films of variousthicknesses, packets were prepared and the oxygen uptake measured at theend of 24 hours.

The data obtained are set out in Table IX below:

TABLE IX Teflon, Mm. 0

mil. consumed (24 hrs.)

Operable Preferred Enzyme system (units) 100-550 200-400 Substrate:

Glucose (gms.) 0. 33-2. 00 0. 75-1. 50 Stabilizer (gIlL/Hll.) 0.10-0.330.15-0.25

Preservative (g) 0. -0; 50 0. -0. 35

Filler (gnL) 0. 10-1. 00 0. 30-0. 60 Buffer (gm.) '0. 01-0. 30 0. 018-0.200 Water (m1.) 1. 00-3. 00 1. 80-2. 20

Particularly preferred and contemplatedfin preferred embodiment of thisinvention is a eompositionotthe following formulation:

Membrane mil polyethylene 0.50 Glucose oxidase units 300 Water ml 1.4Cerelose gm.' 1.3 Citric acid gm .02 Glycerine ml. .60 Sodium gluconategrri. 0.38 Balsa wood (6-9 1b./ cu. .ft.-coarse ground) In thispreferred embodiment of this invention a solution of the sodiumgluconate in the water is mixed with the glycerine and sufficientglucose oxidase to give a potency of 300 units (150 u./ ml.). Thisliquid is placed in one compartment of the polyethylene packet and 1.80g. of a mix of the cerelose, balsa wood and citric acid is placed in theother compartment.

What is claimed is:

1. A deoxygenating body comprising a liquid enzyme system having glucoseoxidase activity and a substrate for said enzyme system, saiddeoxygenating body being enclosed in a water-impermeable, gas-permeablemembrane,

said liquid enzyme system being separated from said substrate byseparating means including a seal rupturable upon application ofpressure to said membrane.

2. A deoxygenating body according to claim 1 wherein said liquid enzymesystem contains a stabilizing material.

3. A deoxygenating body according to claim 1 wherein said liquid enzymesystem contains sodium gluconate.

4. A deoxygenating body according to claim 1 containing in addition tosaid enzyme system and said substrate a buffer material and a filler.

5. A deoxygenating body according to claim 1 wherein said substratecomprises cerelose, said body contains citric acid and ground balsawood.

6. An article of manufacture comprising an envelope of awater-impermeable, gas-permeable membrane having two compartmentsseparated from. each other by a seal rupturable upon application ofpressure to said envelope, one of said compartments containing a liquidenzyme system having glucose oxidase activity and the other compartmentcontaining asubstrate for said enzyme systern.

7. An article of manufacture according to claim 6 wherein said enzymesystem comprises a solution of glucose oxidase in a solution of astabilizing material.

8. An article of manufacture according to claim 6 wherein said enzymesystem comprises a mixture of glucose oxidase, sodium gluconate andglycerine in a Water solution.

9. An article of manufacture according to claim 6 wherein said substratecompartment contains a mixture of a glucose source, a buffering agentand a filler.

10. An article of manufacture according to claim 6 wherein saidsubstrate compartment contains a mixture of cerelose, citric acid, andground balsa wood.

11. An article of manufacture according to claim 6 wherein the liquidenzyme system has a potency offrom to 550 glucose oxidase units,contains from .15 to .25 grams of sodium gluconate and from .25 to .35ml. of glycerine per ml. of solution, and wherein said substratecompartment contains from .75 to 1.5 guns. of cerelose, from .18 to .22gms. of citric acid, and from 0.3 to 0.6 gms. of ground balsa wood.

References Cited in the file of this patent UNITED STATES PATENTS Re.23,523 Baker July 22, 1952 2,245,738 Taylor June 17, 1941 2,758,932Scott Aug. 14, 1956 2,758,934 Scott Aug. 14, 1956 2,765,233 Sarrett eta1. Oct. 2, 1956 2,825,651 Loo et a1. Mar. 4, 1958

1. A DEOXYGENATING BODY COMPRISING A LIQUID ENZYME SYSTEM HAVING GLUCOSEOXIDASE ACTIVITY AND A SUBSTRATE FOR SAID ENZYME SYSTEM, SAIDDEOXYGENATING BODY BEING ENCLOSED IN A WATER-IMPERMEABLE, GAS-PERMEABLEMEMBRANE, SAID LIQUID ENZYME SYSTEM BEING SEPARATED FROM SAID SUBSTRATEBY SEPARATING MEANS INCLUDING A SEAL RUPTURABLE UPON APPLICATION OFPRESSURE TO SAID MEMBRANE.